A serum-free medium (LEP-1) was developed for mouse epidermal keratinocytes, consisting of Eagle's MEM without calcium, with non-essential amino acids and with seven added supplements (hydrocortinsone, O.5 MuM; insulin, 5 Mug/ml; phosphoethanolamine and ethanolamine, 50 MuM each; transferrin, 5 Mug/ml; epidermal growth factor, 5 ng/ml; bovine pituitary extract, 180 Mug of protein/ml). The culture system was dependent for growth on bovine pituitary extract as the only remaining undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was established and used to investigate the action of growth factors, hormones and other supplements on keratinocytes. When the calcium concentration of the medium was raised to 1.0 mM or when 1% FBS was added, the cells underwent terminal differentiation, confirmed by electron microscopy and by immunostaining with anti-keratin antibody. Serum factors were identified as having either stimulatory (albumin) or inhibitory activity (fetuin, crude platelet extract). This finding may explain why keratinocytes could not be carried beyond primary cultures in serum-supplemented media. This mouse keratinocyte culture system in serum-free medium is highly reproducible and is currently used in studies of chemically-induced transformation and gene activation. It was found that the life span of a transformed but non-tumorigenic human prostatic epithelial line (NP-2S/T2) was extended by transfection of the EJ-ras oncongene and that the line containing the oncogene produces a transforming growth factor. In another study, the incidence of metastasis in nude mice of the prostatic adenocarcinoma cell line, PC-3, was increased by isolation of variant cell lines, use of a preferred inoculation site (spleen) and administration of beta-estradiol. The role of oncogenes will be studied in this system, using both the neoplastic and non-transformed human prostate cell lines.